Emergence of new viruses is a continuing threat to our society. Evolution of plus- stranded RNA viruses is frequently driven by RNA recombination, a process that joins noncontiguous RNA segments together, creating novel combinations of genes or regulatory RNA sequences. Recombination is important for RNA viruses to overcome immunity, jump species and change viral pathogenicity in all types of living organisms. Studies on viral RNA recombination are critical to understand the recombination process and the role of specific host genes, whose roles in the recombination process are currently unknown. The investigator will use Tomato bushy stunt virus (TBSV), which is currently the most advanced among viral systems to progress rapidly in understanding the mechanism of RNA recombination and the role of the host genes. The investigator's research opens up a completely new area with the powerful TBSV-yeast model system, which has led to genome-wide screens identifying host genes suppressing RNA recombination for the first time. Additional advantage of the similarity of TBSV replicase proteins to proteins of important pathogens, such as Hepatitis C virus (HCV), dengue virus, West Nile virus and other Flaviviruses and Pestiviruses. Collectively, the major advances with TBSV allow the investigator to conduct experiments on the roles of host factors that are currently only feasible with TBSV, but will open new approaches for studying RNA recombination/evolution for important human pathogens. This proposal focuses on one of the key host factors suppressing RNA recombination. The experiments will dissect the role of Pmr1p Ca2+/Mn2+ pump in viral RNA recombination. Pmr1p is a ubiquitous and conserved protein in eukaryotes. The human homologs of Pmr1p have been shown to play critical roles in genetic diseases, such as Hailey- Hailey Disease and Darier disease. In the absence of Pmr1p, the rate of TBSV RNA recombination is extremely high demonstrating the critical role of the host in viral RNA recombination. In order to characterize the role of Pmr1p in RNA recombination, the investigator will use the advanced genetics tools available for yeast in combination with biochemical approaches. The results obtained in cell-free systems and in yeast model host, will also be confirmed in a native plant host. In addition, in vitro experiments will be conducted with the RdRp of HCV, an important human pathogen, to expand our knowledge on the possible role of Ca2+/Mn2+ pumps in HCV RNA recombination. The following are major strengths of the proposal: (i) Viral RNA recombination is clearly of immense importance for viruses to overcome immunity, jump species and change pathogenicity in all types of living organisms. (ii) The combination of yeast and in vitro systems developed by the investigator is currently the only one in the world suitable for studying the mechanism of host factors involvement in viral RNA recombination. (iii) This research is expected to lead to groundbreaking new discoveries in viral RNA recombination. The highly tractable in vitro and in vivo TBSV system developed by the investigator could prove highly beneficial to studies of other, less amenable RNA viruses. The research holds promise of benefiting society by leading to groundbreaking results in the area of virus recombination/evolution, host-virus interaction and the emergence of new viruses. PUBLIC HEALTH RELEVANCE: Emergence of new viruses is a continuing threat to our society. New plus-stranded RNA viruses emerge frequently due to RNA recombination. Recombination is important for RNA viruses to overcome immunity, jump to new host species or change the severity of symptoms in all types of living organisms. The roles of the host in virus recombination are currently poorly understood due to the lack of tractable virus-host systems. The investigator will use Tomato bushy stunt virus (TBSV), which is currently the most advanced among viral systems to study RNA recombination and the role of the host genes. This proposal focuses on one of the key host factors suppressing RNA recombination by dissecting the role of a Ca2+/Mn2+ pump in viral RNA recombination. The research holds promise of benefiting society by leading to groundbreaking results in the area of virus recombination/evolution, host-virus interaction and the emergence of new viruses.